DETECTION OF INFECTIONS IN BODY FLUIDS

Abstract: Infection in a body fluid is detected by withdrawing a specimen from the body fluid, incubating the specimen or a cell-containing fraction thereof under conditions in which phagocytic white blood cells present in said specimen or said fraction produce superoxide anion (O2<->) and then determining level of the O2<-> in said medium after the incubation.

Description: DETECTION OF INFECTIONS IN BODY FLUIDS FIELD OF THE INVENTION The present invention relates to a method for identifying infection in a body fluid. By one of its preferred embodiments, the invention relates to a method for determining of microbial infection in amniotic fluid. BACKGROUND OF THE INVENTION AND PRIOR ART Microbiological assays are important in clinical practice for the diagnosis of infections. In such assays, the presence of pathological microorganisms in clinical samples, has to be determined. The most widely used microbiological assays involve incubating a biological sample on a growth medium and then determining the occurrence and extent of infection after growth of microorganisms on the growth medium (such assay to be referred to herein, at times, as the "culturing assay"). Such assays, while being relatively accurate, have a major drawback in that they are laborious, require relatively large laboratory space and particularly in that they require relatively long incubation time, about 24-72 hours, and thus do not enable rapid diagnosis of the infection which is required at times for the purpose of provision of immediate treatment. When encountering a case of suspected microorganismal infection, physicians need very often to decide on a treatment even without awaiting the results of the microbiological assay. A particular case in point is the determination of microbial infection in amniotic fluid. Microorganismal invasion into the amniotic fluid increases the risk of prcterm delivery and is also a cause of morbidity of both woman and fetus. Incubating amniotic fluid on a suitable growth medium in order to determine the presence of microorganisms in a sample is a standard clinical practice for the diagnosis of intra-amniotic infection. However, results are only available after 72 hours or even more, which means that a clinical decision regarding the respective patient needs at times to be delayed; at other times an immediate decision regarding treatment has to be made which is then based on incomplete information. One method for rapid detection of microbial invasion into the amniotic cavity is the Gram stain (Romero, R., et al. , Am. J. Obstet. Gynecol., 159, 114, 1988). However, Gram stain provides positive indications of infections in only 50% of cases. Furthermore, the Gram stain method is incapable of detecting mycoplasma infections. Another study (Romero, R., et al. , Am. J. Obstet. Gynecol, 165, 821, 1991) has reported that white blood cell count can be used as a rapid test to diagnose an intra-amniotic microorganismal infection. This study has shown that women which scored positive in a culturing assay, had a significantly higher average amniotic fluid white blood cell (WBC) count than did women which scored negative in such an assay. However, there was a high overlap between the two groups, thus reducing the predictive value of such an assay. Another assay which has been suggested for rapid testing of intra-amniotic infection which has been suggested in the art is the leukocyte — j - esterase test (Egley, C.C., et al. , Λm. J. Obstet. Gynecol, 159, 120, 1988). However, this test showed a sensitivity of only about 50%. It is an object of the present invention to provide a novel assay for the diagnosis of infection in a body fluid. It is an object in accordance with a preferred embodiment of the invention to provide an assay for the diagnosis of an intra-amniotic infection. It is a further object of the invention to provide a kit useful for carrying out the above assays. Further objects of the invention will be apparent from the description below. GENERAL DESCRIPTION OF THE INVENTION In accordance with the present invention a new and highly sensitive method for the determining of microbial infections in body fluids has been developed. By the method of the invention, a body fluid specimen, or a cell-containing fraction thereof is subjected to conditions which will give rise to superoxide anion (0 2 " ) production by such cells. (In the following the term "sample " will be used at times for the sake of conve- nience in order to denote collectively both the body fluid speciment and the cell-containing fraction thereof.) The 0 2 " level is then determined and a level above control indicates a high probability of infection. Furthermore, the level of the Of may serve as indication of the degree of infection. The present invention thus provides a method for detecting a microbial infection in a body fluid, comprising: (a) withdrawing a specimen from a tested body fluid; (b) incubating a medium comprising the specimen or a cell-containing fraction thereof under conditions in which phagocytic white blood cells present in said specimen or said fraction produce superoxide anion (Cy); (c) determining level of the 0 2 " in said medium after the incubation, a level above control signifying a high probability of infection in said body fluid. Examples of body fluids which can be tested by the method of the invention include cerebro-spinal fluid, blood, lymph fluid, urine and other uterine secretions, lubal fluid, follicular fluid, peritoneal fluid, seminal fluid and amniotic fluid, the latter being a preferred embodiment of the invention. The incubated medium may consider of the original withdrawn specimen, at times with some added substances, e.g. such which are required to stimulate the WBC to produce 0 2 ~ ; it may be a mixture of the body fluid specimen with an appropriate liquid medium; or it may be a cell-containing fraction of the specimen suspended in an appropriate medium. A cell- containing fraction may be obtained, for example, by centrifugation, filtration, etc. The control is typically the 0 2 ~ level in a non-infected body fluid. The control may not necessarily be processed simultaneously with the tested body fluid and may typically be an average result obtained from many non-infected body fluids. For control, it is also possible to process another specimen, identical to the tested specimen, under almost identical conditions with the difference befing that either no conditions for the production of 0 2 ~ by the WBC are provided or the inclusion in the medium of an O 2" scavenger, i.e. a substance which either captures or degrades the 0 2 ~ immediately upon production (in this connection see also further below). The WBC present in the sample may be stimulated to produce 0 2 ~ by phorbol myristate acetate (PMA). PMA is typically added at a concen¬ tration of about 100 ng/ml. The incubation period may be in the range of 2-60 mins., depending on the conditions of incubation, e.g. the incubation - _> temperature and whether the invention is performed under agitation, etc. Preferable conditions for incubation are a temperature of about 37°C with agitation. Under such conditions, the required incubation period can be within the range of 10-40 mins., preferably within the range of 20-30 mins. The O, " level can be determined by a number of means known per se. By one example, the presence of anion 0 2 ~ in the sample is determined by colorimetric means, i.e., a change in color; change in other transmission parameters of the fluid, such as transmission in the U.V. range; etc. An example of a colorimetric reaction is one which utilizes the 0 2 " as a redox substrate and which upon reaction with 0 2 ~ gives rise to either disappearance, appearance or a change of a color. An example of such a substrate is acetyl fcrricytochrome C which upon reduction gives rise to a color change from red to pink. Such a color change may be detected visually as well as by means of a spectrophotometcr at a wavelength of 550 nm. Visual detection is generally useful for a qualitative assessment of the occurrence of an infection. Against this, detection by means of a spectrophotomcter, will allow also quantification of the infection degree: optical density (OD) measurement by a spectrophotometer may be convert- ed, as known, per se, to 0 2 ~ levels, and these, once determined, may serve as an indication of the extent of infection. Substrate such as fcrricytochrome C may also be reduced by various other agents which can be present in body fluids. In order to distinguish between a specific reduction (i.e., such which is induced by 0 2 " ) and non-specific reduction (i.e., such which is induced by a reducing agent other than 0 2 ~ ), the level of 0 2 ~ as determined in step (c) may be compared to that obtained in a sample incubated under the same incubation conditions, excluding, however, conditions in which the WBC produce 0 2 " . For example, one test sample may be incubated with PMA, and another, control sample will be incubated under the same conditions but without PMA. Any change in the level of reduction in both cases may then be attributed to the WBC produced Of. Another and preferred way for distinguishing between a specific reduction (as defined above), and a non-specific reduction is to utilize an 0 2 " scavenger, such as the superoxide dismutase enzyme (SOD) (Pick E., et al , J. Immunol Methods, 46, 21, 1981). SOD is an enzyme which captures and degrades Of and therefore in the presence of a sufficient quantity of SOD any reduction otherwise induced by Of will be inhibited. Thus, the reduction measured in the absence of SOD to that obtained in its presence are compared and, any change can then be attributed to the superoxide anion produced by the WBC. The method can in principle proceed in two discrete stages, a first stage of Of production (method step (b)) and a second stage of superoxide level determination (method step (c)). However, by a preferred embodiment of the invention, the two steps are performed simultaneously. By this preferred embodiment, where, for example, a substrate which changes its color upon reduction by (Of) is being used, such a substrate is introduced into the medium together with the sample and the color reaction thus progresses simultaneously with the rise in the Of level in the medium. The entire reaction is preferably performed in a buffer system which comprises isotonic amounts of both NaCl and KG, a buffer, e.g. Hepes buffer, pH 7.4, glucose as well as an amount of MgG 2 , e.g., about 0.4-1.2 mM. The invention also provides a kit comprising the reagents and optionally the hardware required to carry out the above method. An example of such a kit is one which comprises acetyl ferricytochrome C and a substance which can stimulate cells in a sample to secrete the superoxide anion, such as phorbol myristate acetate (PMA). The kit preferably comprises also SOD. The kit may further comprise a medium for carrying out the enzymatic reaction, e.g. a buffer of pH of about 7.4, such as the - 1 - The kit may also comprise one or more vessels, e.g. a microtiter plate for carrying out the assay. Furthermore, the kit may also comprise a color chart to allow a better qualification and at times also quantification assessment of infection. The invention will now be illustrated in the following examples and in the annexed drawing, with reference to the non-limiting, albeit preferred, embodiment of assaying for infections in amniotic fluid. It will no doubt be appreciated by the artisan that the invention also applies, mutatis mutandis, to assaying of infections in other body fluids. BRIEF DESCRIPTION OF THE DRAWING Fig. 1 in the drawing shows the activity of an amniotic fluid sample in reduction of acetyl ferricytochrorne C, without (filled circles - "© ") or with (empty circles "O ") SOD. Example 1 Amniotic fluid was obtained from pregnant women having an intra-amniotic infection. 2 ml amniotic fluid samples were centrifuged at 500 xg for 10 mins, and the pellet was then suspended in 450 μ\ of a buffer (consisting of 137 mM NaCl, 0.8 mM Mg Cl 2 , 5.4 mM KG, 5.6 mM glucose, 10 mMepes, pH 7.4). 50 μ\ of acetyl fcrricytochrome C solution was then added to yield a final acetyl ferricytochrorne C concentration of 150 mM. 3 of 100 μ\ specimens were then placed in wells of microtiter plate. 5 μ\ of a SOD solution, to yield a final SOD concentration of 2.5 ng/ml, was added to one well of each triplicate. The reduction reaction of acetyl ferricytochrorne C was initiated by the addition of 5 μ\ of a PMA solution, to yield a final PMA concentration of 100 ng/ml. The wells were incubated at 37°C and coliemetric readings were made at several time intervals. The reduction of acetyl fcrricytochrome C resulted in a change in color from red to pink, which color change was determined by a spectrophotometer at 550 nm. Exemplary results arc shown in Fig. 1 where filled circles (©) are 5 results from a well which did not contain SOD and the empty circles (O) from a well which contained SOD. These results demonstrate that the generation of superoxide can easily be determined by a simple colorimetric enzymatic reaction. 10 Example 2 Amniotic fluid from 140 pregnant women was tested by the invention assay described in Example 1 (hereinafter "superoxide assay"). Results obtained by the superoxide assay were compared to results obtained by the standard culture assay. Furthermore, at the same time infection was 15 also determined by a count of white blood cells (WBC); the presence of WBC in the sample was determined microscopically following a Wright Giemsa staining procedure (Levy R., et al , Hepatology, 17, 908, 1993). (Positive sure by the WBC assay means a control abut 50 cells/ml). In addition, the occurrence of prctcrm delivery in each of the tested women 20 was also observed. The results which were obtained are shown in the following Table I. Table I __D 30 3D SUBST/TWE SHEET (RUE 26) - 12 " " - positive results; in the case of the superoxide test, this means standard level of superoxide production (above 1.5 nmoles 0 2 7min but below 20 20 nmoles/min) "44 " - very high levels of superoxide production (> 20 nmoles 0 2 7min) __D The above results can be summarized briefly as follows: Of the 140 tested women, there were 112 cases (80%) where showed a correlation between results obtained by the inventive assay and results obtained by the standard culture assay (a correlation meaning that these were either a positive score in both cases or a negative score 30 in both cases). Out of the 140 tested women, 88 showed no sign of infection by the superoxide assay. In all cases where the inventive assay detected no infection, no infection was found also by either one of the two other assays. 3D 52 women scored positive when tested for infection in accordance with the inventive method. Of these 52 cases, in 29 cases (55.7%) scored SUBSTITUTE SHEET ( ULE 26) positive when tested by the culture assay and 41 (78.8%) scored positive by either the culture assay or a WBC count. It should be noted that there was not even one case which scored positive either by the culture assay or by the WBC count, and did not score positive in the superoxide assay. 4. The significance of the method of the invention can be appreciated by the fact that in 10 out of the 12 women which scored positive by the superoxide assay and which scored negative by both the culture assay and by the WBC count, had a prelcrm delivery. Such a preterm delivery could have likely been prevented if the inventive peroxide assay would have been used as the standard for determining infection. The superoxide assay was also compared to the enterase assay (Egley, et al , 1988, supra) and the results obtained by Gram staining (Romero, et al , 1988, supra) (results not shown) and it was demonstrated that the superoxide assay is also more sensitive and accurate than these two assays. SUBSTITUTE SHEET (RU E 25,

Claims: CLAIMS: 1. A method for detecting a microbial infection in a body fluid, comprising: (a) withdrawing a specimen from a tested body fluid; (b) incubating a medium comprising the specimen or a cell-containing fraction thereof under conditions in which phagocytic white blood cells present in said specimen or said fi action produce superoxide anion (c) determining level of the 0 2 " in said medium after the incubation, a level above control signifying a high probability of infection in said body fluid. 2. A method according to Claim 1, wherein said body fluid is amniotic fluid. 3. A method according to Claim 1, wherein the conditions in which phagacytic white blood cells produce 0 2 " , compiiscs the addition of phorbol myristate acetate into the medium. 4. A method according to any one of the preceding claims, wherein the presence of the 0 2 ~ in the medium is determined by colorimetric means. 5. A method according to Claim 4, wherein said colorimetric means comprise a redox substrate which upon reaction with Of gives rise to either disappearance, appearance or a change of color. 6. A method according to Claim 5, wherein said substance is acetyl ferricytochrorne C. 7. A method according to any one of the preceding claims, comprising comparing the level of the tested body fluid 0 2 " determined in step (c), to that of a specimen or cell-containing fraction thereof incubated under the same incubation conditions, excluding, however, the conditions in which white blood cells in the medium produce 0, " . 8. A method according to any one of the preceding claims, comprising comparing the level of the tested body fluid determined in step (c) of 0 2 ~ , to that of a sample incubated under the same incubation conditions in the presence of an 0 2 " scavenger. 9. A method according to Claim 8, wherein the 0 2 " scavenger is superoxide dismutase. 10. A kit for carrying out the method according to any one of the preceding claims.

Inventors: LEVY RACHEL, MAZOR MOSHE, LEVY, RACHEL, MAZOR, MOSHE
Applicants: HELFGOTT & KARAS P C, LEVY RACHEL, MAZOR MOSHE, MOR RESEARCH APPLIC LTD, HELFGOTT & KARAS, P.C., LEVY, RACHEL, MAZOR, MOSHE, MOR RESEARCH APPLICATIONS LTD.
Application year: 1994
Main classification: C12Q1
IPC codes: C12Q1